Volatile anesthetic (VA) agents produce rapidly inducible and reversible states of unconsciousness that are vital for the delivery of general anesthesia, but also cause profound systemic hypotension, depressed respiratory drive, and numerous other deleterious physiological perturbations that increase risks of morbidity or mortality during surgery. Tandem pore (K2P) ion channels are VA responsive potassium leak channels that are fundamental to basic human physiology and suspected mediators of some deleterious effects of VA?s. In this K08 proposal, I will utilize my background in the field of ion channel physiology and biophysics to explore the molecular mechanism by which VAs modulate K2P channels. In Aim1, I will define the molecular determinants of VA binding to K2P channels, utilizing photoaffinity analogs of the haloether VAs isoflurane and sevoflurane. I will identify VA binding sites in two VA sensitive K2P channels (TREK1 and TASK1) and explore the molecular basis for the VA insensitivity of TRAAK K2P channels. Photolabeling results will be verified for functional relevance by introducing mutations at identified anesthetic binding site residues and performing functional studies to assess for altered gating behavior or anesthetic sensitivity. Molecular dynamics simulation guided by photolabeling and functional studies will identify additional residues predicted to contribute to VA binding. In Aim2, I will determine the mechanism by which VA binding alters K2P conformation to effect gating. Single particle cryo-electron microscopy of TREK1 will be utilized to explore the effects of VA?s on K2P conformational state and will produce the first structural characterization of the K2P C- terminal domain known to regulate the modulatory effects of VAs and many other K2P modulators. By utilizing a rapid mixing stopped flow fluorometric assay capable to resolving changes in the kinetics of K2P open pore block by quaternary ammonium ions, I will examine the combinatorial effects of surrounding ionic composition, lipid environment, VAs and other TREK1 modulators on the intracellular pore structure of TREK1, providing functional context to our structural studies. By studying the biophysical basis for the interaction between K2P channels and VA?s, I hope to lay the groundwork for a career translating studies of ion channel structure, function and pharmacology into meaningful clinical interventions that improve the safety of anesthetic care. My K08 research mentor, Dr. Crina Nimigean, is an international expert in the study of ion channel biophysics and is extremely well suited to provide mentorship and guidance during the execution of this research project. The study of ion channel pharmacology and biophysics as they pertain to mechanisms of anesthetic action has been a historical focus of interest in the Weill Cornell Department of Anesthesia and both the departmental and institutional environments provide an outstanding backdrop to enable successful completion of the proposed studies, providing resources, protected research time, and an intellectually enriching environment with numerous available advisors and potential collaborators.